Cholesterol is a naturally occurring substance in the body that is required for normal biological functions. For example, it is used for the synthesis of bile acids in the liver, the manufacture and repair of cell membranes, the production of vitamin D, and the synthesis of steroid hormones. There are both exogenous and endogenous sources of cholesterol. For example, the average American consumes about 450 mg of cholesterol each day and produces an additional 500 to 1,000 mg in the liver and other tissues. Another source is the 500 to 1,000 mg of biliary cholesterol that is secreted into the intestine daily; about 50 percent is reabsorbed (enterohepatic circulation).
Cholesterol circulates in the bloodstream via plasma lipoproteins, which are particles of complex lipid and protein composition that transport lipids in the blood. There are specific kinds of lipoproteins that contain cholesterol, namely low density lipoproteins (LDL), high density lipoproteins (HDL), and triglycerides.
LDL normally carries about 75 percent of the circulating cholesterol. LDL is believed to be responsible for the delivery of cholesterol from the liver, where it is synthesized or obtained from dietary sources, to extrahepatic tissues in the body. The term “reverse cholesterol transport” describes the transport of cholesterol from extrahepatic tissues to the liver, where it is catabolized and eliminated.
As free cholesterol liberated from LDL accumulates within cells, there are three important metabolic consequences. First, there is a decrease in the synthesis of HMG-CoA reductase, the enzyme that controls the rate of de novo cholesterol synthesis by the cell. Second, there is activation of the enzyme acyl cholesterol acyltransferase (ACAT), which esterifies free cholesterol into cholesterol ester, the cell's storage form of cholesterol. Third, accumulation of cholesterol suppresses the cell's synthesis of new LDL receptors. This feedback mechanism reduces the cell's uptake of LDL from circulation.
In contrast, plasma HDL particles appear to play a major role in the reverse transport process by acting as scavengers of tissue cholesterol. HDL is also responsible for the removal of non-cholesterol lipid, oxidized cholesterol, and other oxidized products from the bloodstream. It is hypothesized that high levels of plasma HDL are not only protective against coronary artery disease, but may actually induce regression of atherosclerotic plaque (i.e., see Badimon et al., Circulation 86:(Suppl. III)86-94 (1992); Dansky and Fisher, Circulation 100:1762-3 (1999)).
Currently, an estimated 105 million American adults have undesirable (high) cholesterol levels—namely total blood cholesterol levels of 200 milligrams per deciliter (mg/dL) and higher. Of these, 42 million have cholesterol levels of 240 mg/dL or higher, and are considered a high health risk population. (Centers for Disease Control: National Center for Health Statistics as published by the American Heart Association, Heart and Stroke Statistics—2003 Update. Dallas, Tex.: AHA, 2002).
The very property that makes cholesterol useful in the cell membranes, namely its insolubility in water, also makes it potentially lethal when large amounts of cholesterol are circulating in blood. For example, high cholesterol is commonly associated with an increased risk of heart attack, atherosclerosis and circulatory disorders. In addition, a variety of diseases are caused by disorders of cholesterol catabolism, such as gallstone disease, atherosclerosis, hyperlipidemia and some lipid storage diseases.
Atherosclerosis, for example, is a slowly progressive disease characterized by the accumulation of cholesterol within the arterial wall. Compelling evidence has been submitted regarding the role of oxidized LDL in the formation of artherosclerotic lesions. (Chisolm, Clin. Cardiol, 14:I-25-I-30 (1991)). As LDL becomes oxidized, its properties and mechanisms of interaction with cells are altered extensively. These changes cause the oxidized LDL to act deleteriously at various levels of artherosclerotic lesion development.
Abundant evidence indicates that lowering undesirable cholesterol levels will diminish or prevent atherosclerotic complications. In addition to a diet that maintains a normal body weight and minimizes concentrations of lipids in plasma, therapeutic strategies for lowering cholesterol levels include elimination of factors that exacerbate high cholesterol and the administration of therapeutic agents that lower plasma concentrations of lipoproteins, either by diminishing the production of lipoproteins or by enhancing the efficiency of their removal from plasma. For example, recent studies have shown that taking antioxidants such as vitamin E or beta carotene, reduces an individual's risk of heart attack presumably by preventing the oxidation of LDL (See NY Times, p. A9, cols. 1-6, Nov. 19, 1992).
Additional methods for maintaining a desirable/healthy serum cholesterol levels include the use of cholesterol-lowering agents (i.e., lavostatin, pravastatin, simvastatin, fluvastatin, and atorvastatin). Several trials of the long-term effects of cholesterol-lowering drugs on patients have shown reduced death from and incidence of heart disease. (See Lipid Research Clinics Investigators, Arch Intern Med. 152:1399-1410 (1992)). Although these drugs can produce significant reductions in serum cholesterol, most if not all have undesirable side effects.
Although it has been demonstrated that estrogens have beneficial effects on serum LDL, long-term estrogen therapy has been implicated in a variety of disorders, including an increase in the risk of uterine cancer and possibly breast cancer. Recently suggested therapeutic regimens, which seek to lessen the cancer risk, such as administering combinations of progestogen and estrogen, cause the patient to experience regular bleeding, which is unacceptable to most older women. Furthermore, combining progesterone with estrogen seems to blunt the serum cholesterol lowering effects of estrogen. Concerns over the significant undesirable effects associated with estrogen therapy, support the need to develop alternative therapies for lowering undesirable cholesterol levels, which generate desirable effects on serum LDL but do not cause undesirable effects.
Diabetes, which is often linked with high cholesterol, is a chronic disease that has no cure. Currently, about 18.2 million people or 6.3% of the population in the United States have diabetes. While roughly 13 million have been diagnosed, it is estimated that 5.2 million people are not aware that they have the disease. As the 6th leading cause of death by disease in 2000, diabetes is costing the US health care system an estimated $132 billion annually. National Diabetes Information Clearinghouse, NIH Publication No. 04-3892, November 2003. More serious than the economic costs associated with diabetes are the decrease in quality of life, serious health complications/consequences, and deaths associated with diabetes.
With about 12,000 to 24,000 new cases each year, diabetes is the leading cause of new cases of blindness in adults ages 20-74. Diabetes is also the leading cause of end-stage renal disease, accounting for about 44% of new cases annually. In 2001 alone, approximately 42,800 people initiated treatment for end stage renal disease (kidney failure) because of diabetes. About 60-70 percent of people with diabetes have mild to severe forms of diabetic nerve damage, which, in severe forms, can lead to lower limb amputations. In fact, more than 60% of non-traumatic, lower limb amputations are performed on persons with diabetes. In 2002-2003, about 82,000 non-traumatic, lower limb amputations were performed on persons with diabetes. People with diabetes are 2 to 4 times more likely to suffer a stroke. Moreover, adults with diabetes have heart disease death rates about 2 to 4 times higher than those without diabetes.
Diabetes is a group of diseases characterized by high blood glucose levels, which result from defects in insulin production, insulin action, or both. Because diabetes can remain undiagnosed for years, many people become aware that they have diabetes only after the development of one of its life-threatening complications. Although the cause of diabetes is still unknown, it is well-accepted that both genetics and environmental factors, such as obesity and lack of exercise, are important factors.
One group of diabetes, Type 1 diabetes (or insulin-dependent diabetes mellitus or juvenile-onset diabetes), develops when the body's immune system destroys pancreatic cells that make the hormone insulin, which regulates blood glucose levels. Type 1 diabetes usually occurs in children and young adults; although disease onset can occur at any age. Type 1 diabetes accounts for about 5 to 10 percent of all diagnosed cases of diabetes. Risk factors for Type 1 diabetes include autoimmune, genetic, and environmental factors. Individuals diagnosed with Type 1 diabetes require daily delivery of insulin via injections or pumps.
Another group of diabetes, Type 2 diabetes (or non-insulin-dependent diabetes mellitus or adult-onset diabetes), is a metabolic disorder resulting from the body's inability to make enough, or properly use, insulin. This disease usually begins as insulin resistance, a disorder in which the cells do not use insulin properly, and as the need for insulin rises, the pancreas gradually loses its ability to produce insulin. Type 2 diabetes is the most common form of the disease accounting for 90-95 percent of diabetes. Type 2 diabetes is nearing epidemic proportions, due to an increased number of older Americans, and a greater prevalence of obesity and a sedentary lifestyle.
Gestational diabetes refers to a form of glucose intolerance that is diagnosed in pregnant women. During pregnancy, gestational diabetes requires treatment to normalize maternal blood glucose levels to avoid complications in the infant. A percentage (5-10 percent) of women with gestational diabetes have Type 2 diabetes after pregnancy. Women who have had gestational diabetes also have a 20-50 percent chance of developing diabetes in the next 5-10 years.
Hyperinsulinemia refers to the overproduction of insulin by pancreatic cells. Often, hyperinsulinemia occurs as a result of insulin resistance, which is a condition defined by cellular resistance to the action of insulin. Insulin resistance, as defined above, is a state/disorder in which a normal amount of insulin produces a subnormal biologic (metabolic) response. For example, in insulin-treated patients with diabetes, insulin resistance is considered to be present whenever the therapeutic dose of insulin exceeds the secretory rate of insulin in normal person.
Hypertension has been associated with hyperinsulinemia. Insulin acts to promote vascular cell growth and increase renal sodium retention, among other things. These latter functions can be accomplished without affecting glucose levels and are known causes of hypertension. Peripheral vasculature growth, for example, can cause constriction of peripheral capillaries while sodium retention increases blood volume. Thus, the lowering of insulin levels in hyperinsulinemics can prevent abnormal vascular growth and renal sodium retention caused by high insulin levels and thereby alleviate hypertension.
Impaired glucose homeostasis (or metabolism) refers to a condition in which blood sugar levels are higher than normal but not high enough to be classified as diabetes. There are two categories that are considered risk factors for future diabetes and cardiovascular disease. Impaired glucose tolerance (IGT) occurs when the glucose levels following a 2-hour oral glucose tolerance test are between 140 to 199 mg/dl. IGT is a major risk factor for Type 2 diabetes and is present in about 11 percent of adults, or approximately 20 million Americans. About 40-45 percent of persons age 65 years or older have either Type 2 diabetes or IGT. Impaired fasting glucose (IFG) occurs when the glucose levels following an 8-hour fasting plasma glucose test are greater than 110 but less than 126 mg/dl.
Premature development of atherosclerosis and increased rate of cardiovascular and peripheral vascular diseases are characteristic features of patients with diabetes. Hyperlipidemia is an important precipitating factor for these diseases. Hyperlipidemia is a condition generally characterized by an abnormal increase in serum lipids in the bloodstream and is an important risk factor in developing atherosclerosis and heart disease. For a review of disorders of lipid metabolism, see, e.g., Wilson, J. et al., (ed.), Disorders of Lipid Metabolism, Chapter 23, Textbook of Endocrinology, 9th Edition, (W. B. Sanders Company, Philadelphia, Pa. U.S.A. 1998).
Serum lipoproteins are the carriers for lipids in the circulation. They are classified according to their density: chylomicrons; very low-density lipoproteins (VLDL); intermediate density lipoproteins (IDL); low density lipoproteins (LDL); and high density lipoproteins (HDL). Hyperlipidemia is usually classified as primary or secondary hyperlipidemia. Primary hyperlipidemia is generally caused by genetic defects, while secondary hyperlipidemia is generally caused by other factors, such as various disease states, drugs, and dietary factors. Alternatively, hyperlipidemia can result from both a combination of primary and secondary causes of hyperlipidemia. Elevated cholesterol levels are associated with a number of disease states, including coronary artery disease, angina pectoris, carotid artery disease, strokes, cerebral arteriosclerosis, and xanthoma.
Dyslipidemia, or abnormal levels of lipoproteins in blood plasma, is a frequent occurrence among diabetics, and has been shown to be one of the main contributors to the increased incidence of coronary events and deaths among diabetic subjects (see, e.g., Joslin, E. Ann. Chim. Med. (1927) 5: 1061-1079). Epidemiological studies since then have confirmed the association and have shown a several-fold increase in coronary deaths among diabetic subjects when compared with nondiabetic subjects (see, e.g., Garcia, M. J. et al., “Morbidity and mortality in diabetics in the Framingham population. Sixteen year follow-up study,” Diabetes, 23:105-11 (1974); and Laakso, M. and Lehto, S., “Epidemiology of risk factors for cardiovascular disease in diabetes and impaired glucose tolerance,” Atherosclerosis, 137 Suppl:S65-73 (1998)). Several lipoprotein abnormalities have been described among diabetic subjects (Howard B., et al., “Lipoprotein composition in diabetes mellitus,” Artherosclerosis, 30:153-162 (1978)).
Hyperglycemia, a common feature of diabetes, is caused by decreased glucose utilization by liver and peripheral tissues and an increased glucose production by liver. Glucokinase (GK), the major glucose phosphorylating enzyme in the liver and the pancreatic β-cells, plays an important role in regulating blood glucose homeostasis. Notably, the levels of this enzyme are lowered in patients with Type 2 diabetes (Caro, J. F. et al., Hormone metabolic Res., 27; 19-22, 1995) and in some diabetic animal models (Barzilai, N. and Rossetti, L. J. Biol. Chem., 268:25019-25025, 1993).
As supported above, virtually every major organ system in the body is damaged by diabetes. Complications can include blindness, kidney failure, heart disease, stroke, amputation of extremities, loss of nerve sensation, early loss of teeth, high-risk pregnancies and babies born with birth defects. Currently, insulin injection is the only treatment method available for the over 1.5 million Type 1 diabetics and becomes the eventual course of treatment for many of the more than 16 million Type 2 diabetics in the United States. Treatment of Type 2 diabetes usually consists of a combination of diet, exercise, oral hypoglycemic agents, e.g., thiazolidinediones, and in more severe cases, insulin. However, the clinically available hypoglycemic agents can have side effects that limit their use, or an agent may not be effective with a particular patient.
In the case of Type I, insulin is usually the primary course of therapy. In spite of the early discovery of insulin and its subsequent widespread use in the treatment of diabetes, and the later discovery of and use of sulfonylureas, biguanides and thiazolidinediones, such as troglitazone, rosiglitazone or pioglitazone, as oral hypoglycemic agents, the treatment of diabetes remains less than satisfactory. Nutritional therapies that positively impact glucose uptake in the face of insulin insufficiency would have a major impact on the long term treatment costs associated with diabetic care.
Adiponectin or Acrp30 (Hu, E. et al, “AdipoQ is a novel adipose-specific gene dysregulated in obesity,” J. Biol. Chem., 271:10697-10703 (1996)) is an adipocyte-derived hormone with multiple biological functions. It has been reported that obesity, Type 2 diabetes and coronary heart disease are associated with decreased plasma adiponectin levels, and that adiponectin may have putative anti-atherogenic properties in vitro (Ouchi, N. et al, “Adipocyte-derived plasma protein, adiponectin, suppresses lipid accumulation and class A scavenger receptor expression in human monocyte-derived macrophages,” Circulation, 103:1057-1063 (2001); Yokota, T. et al, “Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages,” Blood, 96:1723-1732 (2000)).
It has also been reported that an acute increase in circulating levels of Acrp30 lowers hepatic glucose production (Berg, A. H. et al, “The adipocyte-secreted protein Acrp30 enhances hepatic insulin action,” Nat. Med., 7:947-953 (2001); Combs, T. P. et al, “Endogenous glucose production is inhibited by the adipose-derived protein Acrp30,” J. Clin. Invest., 108:1875-1881 (2001)). Moreover, it has been reported that globular Acrp30 increases fatty acid oxidation in muscle, and causes weight loss in mice (Fruebis, J. et al, “Proteolytic cleavage product of 30-kDa adipocyte complement-related protein increases fatty acid oxidation in muscle and causes weight loss in mice,” Proc. Natl. Acad. Sci. USA, 98:2005-2010 (2001)). Further, it has been reported that treatment with adiponectin consisting solely of the globular domain (globular adiponectin or gAd) increases fatty acid oxidation in muscle, thereby ameliorating insulin resistance in lipoatrophic mice and obese mice, while treatment with full-length adiponectin also ameliorates though less than with gAd (Yamauchi, T. et al, “The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity,” Nat. Med., 7:941-946 (2001)).
Recently it has been reported that adiponectin acutely activates AMP kinase (AMPK) in skeletal muscle, thus stimulating fatty acid oxidation and glucose uptake (Yamauchi, T. et al, “Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase,” Nat. Med., 8:1288-1295 (2002)), and that adiponectin chronically activates PPARα, resulting in increased fatty acid oxidation but reduced tissue TG content in the muscles, with these effects being greater with gAd than with full-length adiponectin (Yamauchi, T. et al, “Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis,” J. Biol. Chem., 278:2461-2468 (2002)). Interestingly, in the liver full-length adiponectin alone acutely activates AMPK, causing a reduction in gluconeogenesis-associated molecules and stimulating fatty-acid oxidation, and moreover full-length adiponectin alone chronically activates AMPK, stimulating fatty-acid oxidation and reducing tissue TG levels in the liver. All these changes serve to enhance insulin sensitivity in vivo (Yamauchi, T. et al, Nat. Med., 8:1288-1295 (2002); Yamauchi, T. et al, J. Biol. Chem., 278:2461-2468 (2002)).
The findings above suggest adiponectin's potential involvement in obesity, cardiovascular disease, and diabetes. Production and circulating adiponectin concentrations are suppressed in obese mice and humans (Hu, et al., J. Biol. Chem., 271:10697-107032 (1996); Arita, et al., “Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity,” Biochem. Biophys. Res. Commun., 257:79-83 (1999)). Low plasma levels of adiponectin may be a risk factor in coronary heart disease and concentrations are also significantly reduced in Type 2 diabetes (Ouchi, et al., Circulation. 100:2473-2476 (1999); Hotta, et al., Diabetes. 50:1126-1133 (2001)). The ability of adiponectin to lower glucose and reverse insulin resistance suggests that it may 30 have application as a diabetes drug (Yamauchi, et al., Nat. Med. 7:941-946 (2001); Berg, et al. Nat. Med. 7:947-953 (2001)). Furthermore, a proteolytically cleaved fragment of adiponectin was shown to cause weight loss in obese animals (Fruebis, et al., Proc. Natl. Acad. Sci. USA. 98:20(15-2010 (2001)). This protein directly or indirectly affects at least four cell types. Adiponectin modulates NF-.kappa.B mediated signals in human aortic endothelial cells, presumably accounting for their reduced adhesiveness for monocytes (Ouchi, et al., “Adiponectin, an adipocyte-derived plasma protein, inhibits endothelial NF-kappaB signaling through a cAMP-dependent pathway,” Circulation, 102:1296-1301 (2000)). The protein suppresses differentiation of myeloid progenitor cells and has discrete effects on two monocyte cell lines (Yokota, Blood. 96:1723-1732 (2000)). Adiponectin may also induce metabolic changes in hepatocytes (Yamauchi, et al., 2001; Berg, et al. 2001).
Insofar as is known, cysteamine compounds have not been previously reported as being useful in modulating biological factors such as adiponectin levels and blood uric acid levels to treat abnormally functioning metabolism (i.e., glucose or lipid metabolism).